Grantee Research Project Results

2011 Progress Report: Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered Plants

EPA Grant Number: R834824
Title: Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered Plants
Investigators: Baumert, Joseph L , Goodman, Richard E. , Peterson, Daniel H
Institution: University of Nebraska at Lincoln
EPA Project Officer: Aja, Hayley
Project Period: September 15, 2010 through September 14, 2013 (Extended to September 14, 2014)
Project Period Covered by this Report: September 15, 2010 through September 14,2011
Project Amount: $423,546
RFA: Approaches to Assessing Potential Food Allergy from Genetically Engineered Plants (2009) RFA Text | Recipients Lists
Research Category: Human Health

Objective:

The proposed research focuses on the development of a more reliable, practical and predictive animal model that can be used to evaluate the allergenic potential of proteins introduced into genetically engineered plants. The objectives of this research project are to: (1) evaluate sensitization responses in germ-free mice and mice having different defined intestinal microflora, to orally presented purified proteins (potent allergen, peanut Ara h 2; moderate allergen, egg-white lysozyme and non-allergenic soybean lipoxygenase); (2) evaluate the importance of the food matrix (peanut, egg-white and soybean) on sensitizing potential of pure proteins; and (3) test for differences in absorption (serum concentrations) of sensitizing proteins (Ara h 2, lysozyme and soybean lipoxygenase) in (1) and (2), as a potential source of differences in allergic sensitization.

Progress Summary:

This progress report covers research activities completed or in progress during the first year (September 2010 through September 2011) under the EPA STAR Grant # RD834824. Funding was received on October 30, 2010, and research commenced at that time. A summary of the research activities for this period are provided below:

1. Protein Purification:

Purified proteins, including bovine beta-lactoglobulin (BLG), soybean lypoxygenase, hen’s egg lysozyme (HEN), and peanut Ara h 2 protein have been acquired from various sources, and the purity has been assessed using SDS-PAGE under non-reducing conditions and with specific antibody immunoblotting analysis where antibodies were available. A representative evaluation of the purity of the bovine BLG used for subsequent oral sensitization and challenge studies of mice is provided in Figure 1. The BLG acquired from commercial sources was found to have >90% purity as observed by non-reducing SDS-PAGE. IgG immunoblot analysis using rabbit anti-BLG IgG antibody, followed by a goat anti-rabbit IgG antibody labeled with HRP, was used for visualization of the BLG proteins. You will note that a light band at approximately 37 kDa was observed in the IgG immunoblot. Because the gel was run under non-reducing conditions, we believe the light bands are due to the formation of a dimer of BLG. We have excised these bands and have sent them off for tandem mass spectrometry analysis to identify the sequence of the protein band. The data were not available at the time that this report was drafted. Small quantities of peanut Ara h 2 protein and soybean lipoxygenase have been acquired, and additional quantities of each protein are being purified in Dr. Baumert’s laboratory.

Figure 1. Evaluation of the purity of commercially available bovine beta-lactoglobulin (BLG) using non-reducing SDS-PAGE and IgG immunoblotting. 1A. Non-reducing SDS-PAGE analysis of BLG. 1B. IgG immunblot analysis of BLG using rabbit anti-BLG antibodies. Lane M – molecular weight marker in units of kDa. Lane A – nonfat dried milk (5 ug protein load). Lane B – whey protein (5 ug protein load). Lane C – BLG (lot 1, 3.3 ug protein load). Lane D - BLG (lot 1, 1.6 ug protein load). Lane E – BLG (lot 2, 3.3 ug protein load). Lane F - BLG (lot 2, 1.6 ug protein load).

2. ELISPOT Method Development for Detection of HEL-Specific Ig Secreting B Cells:

ELISPOT assay development for the detection of HEL specific IgE and IgG1 secreting B cells isolated from the lamina propria and splenocytes of C57BL/6 mice mono-associated with B. fragilis was initiated during year 1 of this project. Mice were either gavaged with 1 mg HEL+ 10 ug cholera toxin (CT) adjuvant 2 weeks prior to euthanization or sensitized through IP injection with the same dose of HEL + CT. Lamina propria lympocytes or splenocytes were isolated by removal of the epitheleal cells (aided by removal of free calcium), followed by digestion of minced intestinal tissue with a mixture of collagenase and dispase. 1 x 10⁶ cells were added to each well of a Millipore Multiscreen HA plate. 1 ug of HEL per well was coated followed by 24 hours of culture with the isolated lymphocytes. HEL specific IgE and IgG1 secreting B cells were visualized by use of goat anti-mouse antibody labeled with HRP. Spots were developed using a Nova Red substrate kit, scanned and then counted using a C.T.L. ELISPOT reader coupled with ImmunoSpot 5 software. Results of the development of the ELISPOT for detection of HEL specific IgE and IgG secreting B cells have been variable to this point. No or limited numbers of HEL specific IgE or IgG1and IgG secreting B cells have visualized with mice that were sensitized with HEL via oral gavage from either the lamina propria or the splenocytes. These results indicate that ELISPOT detection of specific B cells from the lamina propria lymphocytes and splenocytes of HEL sensitized mice may not work for this particular allergenic protein as it may not be eliciting a robust immune response. We plan to expand this research to include bovine BLG and the peanut allergen Ara h 2, which have been shown to be potent sensitizers for allergy.

3. Development of a Sensitized Mouse Model for Allergy Assessment of Proteins:

Development of a mouse model that can be used for allergy assessment of proteins was initiated by identifying conventional mice strains that would potentially make good models for sensitization before moving on to using these stains in the germ-free or mono-associated mouse models. We anticipate that the germ-free or mono-associated mice will provide a more robust sensitization model; however, due to the expense and care of these mice, it is advantageous to first identify a group of conventional mouse strains that may give a moderate sensitization response. Pilot experiments using conventional C3H/HeN and C57BL/6 mice sensitized with BLG and or whey protein are ongoing.

Future Activities:

Protein purification and characterization/identification will continue in year 2 of the grant to obtain sufficient quantities of peanut Ara h 2 and soybean lipoxygenase if needed. The addition of the AKTA Avant FPLC instrument in Dr. Baumert’s laboratory will aid in increasing the efficiency and repeatability of protein purification from natural food sources. All purified proteins will be analyzed using SDS-PAGE analysis, immunoblot analysis when antibodies directed against the protein of interest are available, and confirmatory tandem mass spectrometry analysis will be conducted. The purified proteins will be used for oral sensitization of conventional, germ-free and mono-associated mice of various strains. Breeding pairs of germ-free C3H/HeN mice will be purchased from INRA in France (from Dr. Sylvie Rabot). These mice will be transported to our facilities at the University of Nebraska at Lincoln (UNL) at the beginning of 2012, allowed to acclimate for 3-4 weeks, and bred to develop a germ-free line of C3H/HeN mice at UNL. Once a suitable population of germ-free mice are developed (anticipated by July 2012), sensitization experiments on the germ-free mice will commence using BLG, Ara h 2, HEL, and soybean lipoxygenase as sensitizing proteins. A portion also will be mono-associated with B. fragilis and evaluated for sensitization and allergic response to the outlined proteins. Work on the B cell ELISPOT will continue to evaluate this methodology to detect specific IgE secreting B cells from lymphocytes of mice sensitized to Ara h 2, BLG, and soybean lipoxygenase. As the mice from these experiments are euthanized, sera will be collected to determine if digestion resistant peptides of these allergenic proteins can be measured with both ELISA methods and by tandem mass spectrometry as outlined in Objective 3 of the proposal.

Journal Articles:

No journal articles submitted with this report: View all 10 publications for this project

Supplemental Keywords:

Anaphylaxis, antibody, GM, risk assessment, sensitive population, human health risk, food allergenicity, oral allergy syndrome, allergic sensitization;

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.